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Comments on Primary Papers and News

The paper by Holmes et al. examines pathology and cognition of eight patients from the AN1792 Aβ vaccination trial. Despite the suspension of this trial in 2002, the patients continued to be followed clinically. Two patients showed almost complete removal of amyloid in the brain. The important finding of the current report is that cognitive decline was identical to placebo-treated patients despite the pronounced removal of amyloid. While these data contrast with the many mouse studies showing cognitive improvement and indeed suggest a more limited role for Aβ in the progression of Alzheimer disease, extensive speculation from such a small cohort should be avoided. In contrast to the current report, the 2003 report from Hock et al. showed slowed cognitive decline in a group of 30 patients over a year following treatment; however, this was correlated with a modified antibody titer; the TAPIR assay (tissue amyloid plaque immunoreactivity; the ability of circulating antibodies to bind to amyloid plaques on tissue) (Hock et al., 2003). In the current study the authors suggest several scenarios for the lack of clinical efficacy: 1) amyloid plaques initiate but do not maintain progressive neurodegeneration, 2) very slow plaque removal, 3) inability to remove oligomeric Aβ, and 4) overactivation of the innate immune system.

An important effect of immunization that has not been reported on in the current study is cerebral amyloid angiopathy (CAA) and microhemorrhage. It has been shown that passive immunotherapy increases CAA in transgenic mice (Wilcock et al., 2004) and causes increased incidence of microhemorrhage (Pfeifer et al., 2001, Wilcock et al., 2004, Racke et al., 2005). We also reported that these adverse events occurred with active vaccination (Wilcock et al., 2007). Indeed, the authors of the current report use CAA and Aβ accumulation around capillaries as histopathological factors used to determine the degree of amyloid clearance. It also seems the microhemorrhage occurrence will be difficult to overcome. The recent report from Schroeter et al. showed that even low doses of antibody, which were associated with essentially no amyloid removal, resulted in an apparent subtle increase in microhemorrhage (Schroeter et al., 2008; control mice had no animals with microhemorrhage rated 2 or 3 while the lowest dose of 3D6 had three mice rated 2 or 3). Accumulation of CAA and associated microhemorrhage likely contributes significantly to the clinical progression of disease. Additionally, as the authors suggest, a change in inflammatory state could certainly contribute to further cognitive decline. Recent data show that the inflammatory profile of Alzheimer’s and transgenic mouse brain is highly complex (Colton et al., 2006). It is likely that Fcγ receptor activation affects the inflammatory state.

These data highlight the significant differences between human and mouse studies. Since neurodegeneration is not abundant in the majority of mouse models, it has not been possible, to date, to study this. It is likely that while amyloid may initiate the cascade, neurodegeneration may be self-perpetuating and neuroprotection may also be critical for successful anti-amyloid therapeutics. It has been suggested that passive immunization will overcome some of the limitations of active vaccination, and we certainly eagerly anticipate the data from Elan’s passive immunization trial of bapineuzumab.

Our study was a six-year follow-up of patients in the original Elan AN1792 study of active immunization of AD patients with full-length Aβ42 peptide. We have confirmed that Aβ immunization can result in plaque removal from the AD brain. The extent of plaque removal is quite variable—ranging from no demonstrable plaque removal to essentially complete removal of plaques from the brain. The extent of plaque removal correlated at least to some extent with the titers of antibodies to Aβ in the serum. Two patients had almost complete removal of plaques from the brain, and yet they still had a progressive decline in cognitive function to profound end-stage dementia shortly before they died. All patients who had postmortem neuropathology had extensive tangles—Braak stages V/VI, consistent with AD. Although our findings are based on small numbers of patients, they seem to demonstrate that the presence of plaques is not a prerequisite for progressive cognitive impairment in AD.

We suggest a number of possible explanations for our findings:

1. The presence of Aβ plaques is required to initiate, but not to maintain the progressive neurodegeneration in AD.

2. Amyloid plaques are an epiphenomenon, and extracellular soluble/oligomeric or intraneuronal forms of Aβ are responsible for the neurodegeneration in AD.

3. Immunization activates microglia, which may be beneficial (by removing plaques) but at the same time neurotoxic.

4. The plaques could have been removed shortly before the patients died, after their cognitive function had declined—this seems rather unlikely.

A major driver of the immunization strategies currently in clinical trials has been to avoid a T lymphocyte reaction in the belief that this is what underlay the side effect noted in the second study of AN1792. Passive immunization, in particular, should theoretically not be able to provoke a T cell response and has the additional benefit that the bioavailability of the antibodies can be controlled. However, it is not clear by what mechanism lymphocytes in the leptomeninges, identified in patients with the side effect, can cause changes in the cerebral white matter (a consistent feature on brain imaging of the affected patients). An alternative explanation for the side effect is that it was due to disaggregation and solubilization of plaque Aβ which then tracks to the cerebral vasculature, increasing the severity of cerebral amyloid angiopathy (CAA). We know from previous studies that severe cerebral amyloid angiopathy is associated with abnormalities in the white matter. Interestingly, the new information from the current Elan trial of passive immunization (bapineuzumab) seems to be showing evidence of white matter abnormalities which are occurring more frequently in patients with ApoE4—known to be associated with more severe CAA.

On the basis of our findings, we would predict that other immunization protocols (e.g., passive immunization and active immunization with truncated versions of the Aβ peptide) will also be effective in clearance of plaques. A number of current studies have before and after immunization in vivo plaque imaging, for example, with PIB, built into their design. We would predict that these will demonstrate plaque clearance following immunization. However, on the basis of our findings, we would speculate that plaque removal will not correlate well with any changes in cognitive function.

It is possible that some of the new immunization protocols will have a different balance of effects on the different forms of Aβ (e.g., plaque, soluble, oligomeric, intraneuronal) and may therefore have different effects on cognitive function. One of the approaches being trialled involves passive immunization with an Fc-truncated antibody, and this may have the potentially beneficial effect of not provoking microglial activation.

Using immunization as prevention rather than treatment would likely avoid these complications which seem to be due to the presence of substantial quantities of Aβ already being present in the brain. On the basis of the animal studies, immunization at a young age can prevent the formation of plaques in later life. Of course, we don’t yet know if this can be done safely in humans—we don’t know the physiological function of Aβ and if immunization might interfere with this function. A study to determine if Aβ immunization at a young age could prevent the development of AD later in life would be the ultimate test of the Aβ hypothesis.

The Next Phase: Prevention. Where Do I Sign Up?
The Aβ vaccination strategy failed because it was not used early enough in the course of the disease.

Come again?

We know this apparently because Aβ oligomers, which are artifacts of ultracentrifugation, when injected into the ventricles of mice, cause mice to navigate water mazes poorly, and press levers inappropriately. We know this because when hippocampal slices are bathed in a suspension of the artifact, they demonstrate electrophysiological abnormalities. And we know this because transgenic mice, which are engineered to overproduce Aβ, and then administered antibodies against it, improve in their ability to navigate water mazes and press the appropriate levers.

We apparently also must set aside the ad hoc revisions and contortions of the amyloid cascade hypothesis over the years (1-3), and the plethora of problems with experimental AD models, from lack of cognitive dysfunction, to lack of neuronal loss, to necessity of multiple mutations, to hyperphysiologic production of a target protein. We set this aside because we apparently now know that synaptic damage, a process never directly assessed, and which probably has the same specificity as gliosis, is the pathological substrate for this laboratory artifact in AD (1).

So a strategy, founded in the analysis of a pathological lesion (once said to be toxic and now discarded as a distraction, except of course for the two subjects who found to be “cleared” of plaques at autopsy), based on an ad hoc modification of a hypothesis that a laboratory artifact specifically causes nonspecific damage that has never been analyzed directly, verified in a transgenic mouse construct that generally does not lose neurons, and which was tested and failed in human disease subjects, must now be used on normal people. Where do I sign up?

The recent follow-up to the AN1792 study by Holmes et al. is a thought-provoking study that reinforces but certainly does not prove speculation by many in the field, including myself (Golde, 2006; Golde, 2003), that therapeutic targeting of Aβ may have limited impact on the clinical disease (Golde, 2006; Golde, 2003). Because of the small number of subjects and the unknown possible untoward consequences of an active vaccination targeting an auto-epitope, I think that this data is simply provocative but certainly not definitive.

I have often used the analogy that anti-Aβ therapy for AD is analogous to treating patients whose coronary arteries are 99 percent clogged with a statin and hoping for a clinical effect. These new data raise the possibility that anti-Aβ immunotherapy is more like trying to treat somebody with massive myocardial contraction deficits following multiple MIs with a statin and a bypass. So much damage has been done that targeting the trigger, by itself, is simply too little too late. Indeed, we would not approach the treatment of a patient in severe cardiac dysfunction that has resulted from multiple MIs as a result of long-standing atherosclerotic disease with a statin alone. It simply is not going to work, though it might have some benefit in combination with other therapeutic agents.

Though a small and vociferous group of colleagues are publicly using such data to refute the role of Aβ aggregation in AD and thus indirectly attempting to invalidate it, Aβ or Aβ aggregates, as a target, I think a more parsimonious approach and one discussed to an extent by the authors is to really think carefully about these data and how we as a field might modify our approach to AD therapy and research based on such studies. Although there are numerous potential implications of these data, I will limit myself to a few issues that I see as most important. Obviously, the following comments may be tempered somewhat by any future demonstration of efficacy in Phase 3 studies of anti-Aβ therapies, but I think they will likely hold even in that event.

From a basic research point of view, this ups the ante on two critical issues.

In order to enable better preclinical studies, we still need better animal models of AD that fully recapitulate all the features of the human disease—especially neuronal loss. Given that this appears very difficult to do in APP mice, we probably need to consider looking at other species. Indeed, this report suggests the AN1792 trial appears to have “worked” in humans as it did in mice. Of course, APP mice are good models of Aβ deposition but not real models of AD. If we had a complete animal model of AD, we might be better able to evaluate therapeutic paradigms for impact on neurodegeneration. Tau mice might be better predictors for effects on neuronal loss, but obviously aren’t much use for testing anti-Aβ therapies. Hopefully they will be predictive of clinical outcomes when novel anti-tau therapies are moved into the clinic.

We need a real understanding of why neurons die in AD, and we need to identify additional therapeutic targets that will protect or restore neuronal function. Indeed, though my own research is Aβ-centric, I believe it is of paramount importance to identify targets beyond Aβ and, for that matter, tau. I think that it is more important to explicitly state that we need additional targets than to try and invalidate current ones.

From a clinical perspective, I think this reinforces our need to figure out how to prophylactically treat AD. We need to directly confront and overcome the challenges that distinguish therapeutic trials from prevention trials. We also need to figure out whether a trial of MCI of the AD type to AD conversion is really a prevention trial or just a very early therapeutic trial. Current predictive AD biomarker initiatives will certainly help to frame and define some aspects of the problem in more detail, but we also need to find common ground on how to actually execute a prophylactic trial that is economically feasible, ethical, and appropriately powered. Such trials will almost certainly require the joint efforts of academic, government, and commercial sectors, and of course, “safe agents.” Indeed, the true test of the Aβ “aggregate/amyloid” hypothesis of AD is a trial to prevent Aβ deposition in humans, not a therapeutic treatment of patients with clinical symptoms.

On a final, more technical note, following the initial report (Nicoll et al., 2003) of plaque clearance in one patient, I was less than convinced that there was clearance. The new data do make me more convinced. However, I would like to see some rigorous biochemical analysis of Aβ levels in the brains of these subjects. Even in mouse models, “plaque loads” seem to overestimate reductions in Aβ as compared to biochemical measures. I am also struck by what appears to be patchy clearance. I find it hard to rationalize how patchy clearance can occur with an antibody-mediated mechanism and wonder whether cellular immune responses play some role in the actual clearance.

This report is an interesting follow-on from a case report that showed evidence of Aβ plaque removal following immunization with the Elan/Wyeth AN1792 Aβ vaccine (Nicoll et al., 2003). Holmes and coworkers (2008) now extend the findings of the original case report to eight additional cases, which demonstrated varying degrees of histological evidence of Aβ plaque clearance. What I found most interesting about this report is that, even within this relatively small sample, the cases that had the most prominent (so-called “very extensive”) evidence of Aβ plaque removal also had the highest Aβ antibody titers. This further cements the relationship between Aβ-directed immunity and plaque clearance, which has now been observed by us and by many others in AD mice.

There are a few issues that I’d like to comment on. I find it noteworthy that seven out of eight cases had MMSE scores of zero when last screened. The authors point out that these were “end stage” AD cases—and judging from the MMSE scores, that’s an understatement. I agree with Todd Golde that AD immunotherapy in this small, severely affected cohort is not a robust test of the amyloid cascade hypothesis in humans. But, I don’t believe that this detracts at all from the provocative nature of the findings, and from the message to keep an open mind and to critically consider the etiological contribution of Aβ to AD. More than likely, what these data are telling us is that there is a cutoff beyond which severe neuronal damage/loss has already occurred, and removing Aβ from the equation will have little if any effect clinically. This has prompted a number of researchers to conclude that prevention by immunotherapy is a more viable strategy. That may be true, but when should vaccination be initiated—five, 10, 20, or more years before symptoms manifest? Also, what biomarkers should be used to determine those at risk: APOE genotype, CSF Aβ, CSF tau, plasma Aβ? At this stage, a preventative Aβ vaccine seems much more viable for the It is unfortunate that an adjuvant-alone (placebo) treatment group could not be evaluated side-by-side with the AN1792-treated cases, and that historical non-vaccinated AD cases had to be used as controls. It is possible that the inflammatory side effects of the Th1-biasing vaccine adjuvant (QS-21) negatively impacted cognitive function and/or survival independently of the synthetic Aβ42 peptide. Along those lines, the authors comment that “only one patient had clinical features of meningoencephalitis….” Did the authors evaluate CD4+ T cells in these vaccinated cases, and if so, were they present in greater quantity than in the historical non-vaccinated AD cases?

In summary, this paper represents a timely, thought-provoking examination of the clinical and pathological correlates of Aβ vaccination. As we move forward in this exciting time of AD therapeutics, it will be important to view the results of such clinical trials with open eyes and without bias toward whichever AD pathogenic hypothesis we hold close to our hearts.

It is worth reading the comments on this paper that have already been posted on Alzforum. This thorough analysis of the long-term effects of the (foreshortened) Elan Aβ-immunization trial (AN1792) in the U.K. is both sobering and, to the BAPtists among us, a bracing challenge. Why, if Aβ plaques are being removed, does cognition continue to deteriorate in immunized AD patients? Both Holmes et al. and the accompanying commentary by St. George-Hyslop and Morris nicely summarize the potential reasons for this disappointment, from the technical (too few subjects to draw firm conclusions) to the mechanistic (e.g., if dementia has already set in, the treatment is too late, or it is necessary to target Aβ oligomers).

These comments should be taken seriously, as they encapsulate key issues that must be addressed if the Aβ cascade hypothesis (or at least the future of immunization therapy) is to survive this trial. To the opponents of the Aβ cascade hypothesis, it might seem that we Aβ stalwarts run the risk of straining a hand-waving muscle right now, but the evidence supporting a primary role of Aβ in disease pathogenesis remains considerable and compelling. Perhaps, as St. George-Hyslop and Morris say, a pluralistic approach will be necessary to address the complex degenerative process, but I believe that a monotherapy eventually will emerge from a deeper understanding of the disease process, particularly in the early stages of AD.

The future of the immunization approach to AD (or of any disease-modifying approach, for that matter) may well lie in prevention. But who will run the lengthy and expensive trials that are needed to determine whether it will work? And will the resolve to test preventive measures be weakened by the failure of therapeutic trials conducted long after the disease has begun to take its toll on the subjects?

This paper is a jarring wake-up call to all Alzheimer disease investigators that placed all their research marbles in the amyloid hypothesis basket, as the clinical pathological findings suggest serious rethinking of the Aβ42 vaccination approach. Based on this report and the mounting evidence from Aβ vaccination trials spoken about at the ICAD meeting, it is becoming clear that an amyloid vaccination mono-therapeutic approach to AD treatment is simply not the sole answer. It can be argued that adding more subjects to the Holmes et al. study is appropriate for further clarification, but both clinical trial and neuropathologic studies of the brain of folks who have come to autopsy with mild cognitive impairment (MCI) provide extensive evidence that amyloid is not a strong correlative of cognitive decline (Mufson et al., 1999; Forman et al., 2005).

Data derived from our ongoing clinical molecular pathologic investigations of MCI using the cholinotrophic basal forebrain system as a model for neuronal selective vulnerability has shown that these neurons display a myriad of biochemical and molecular alterations, which appear to be unrelated to amyloid deposition (Counts and Mufson, 2004). For example, cholinergic neurons are simultaneously undergoing re-expression of cell cycle markers, alterations in neurotrophic support and the ratio of tau epitopes but not changes in APP or presenilin expression. The molecular signature of these neurons is commensurate with a hypothesis related to multiple cellular and connectivity-based dysregulation, which probably begins several decades before the onset of clinical symptoms. What initiates neuronal dysfunction remains unknown, and merits serious research in relevant animal models as well as in well-characterized postmortem human brain tissues. In this regard, it would be of interest to examine the molecular pathology of the cholinergic basal forebrain (CBF) neurons in the same vaccine treated brains examined by Holmes et al. to determine whether amyloid removal from cortical and hippocampal parenchymal projection sites of the CBF neurons rejuvenates these cell bodies.

To anyone who has ever examined the brain of a patient with AD, it is evident that the disease is not simply an amyloidosis. AD is a multi-neuronal system disconnection syndrome of unknown etiology, with pronounced selective cell loss, synaptic dysfunction, atrophy, vascular pathology, tau pathology, in addition to intracellular Aβ disturbances and extracellular amyloid deposition, among other problems that may yet be discovered. It is not our intention to advocate any singular hypothesis of AD, rather to suggest that other treatment approaches and modalities should be pursued with a solid federal and private funding base in addition to amyloid-based clinical trials. An effective treatment will ultimately be a poly-pharmaceutical approach that targets both mechanisms underlying neurodegeneration as well as symptoms of cognitive decline until the etiology of AD is revealed.